Application of tea tree CsTPR38 gene in regulation and control of leaf tooth density of tea tree

Abstract

The invention relates to application of a tea tree CsTPR38 gene in regulation and control of tea tree leaf tooth density, and belongs to the technical field of gene engineering. The complete sequence of the tea tree CsTPR38 gene is obtained by cloning the sequence of the tea tree CsTPR38 gene. Then, the CsTPR38 is subjected to bioinformatics analysis, GUS reporter gene expression is started by constructing a plant expression vector and connecting a target fragment, and a recombinant plant vector is used for genetic transformation of arabidopsis thaliana, so that the function of the CsTPR38 is verified. According to the CsTPR38 gene of the tea tree, the character of a CsTPR38 transgenic line is analyzed, the leaf tooth density is measured, it is found that the CsTPR38 gene of the tea tree can regulate and control the leaf tooth density, and a foundation is laid for breeding new varieties through the CsTPR38 gene.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to tea plants. CsTPR38 Application of genes in regulating leaf tooth density in tea plants. Background Technology

[0002] Tea, as one of the world's three major beverages, possesses high economic and cultural value. The quality of tea is related not only to economic benefits but also to its nutritional value. "Quality" refers to the comparative grade of tea. For example, there are superior and inferior black teas; there are high-grade and low-grade green teas. "Quality" refers to the comprehensive reflection of both appearance and internal characteristics. The quality of tea is a concrete manifestation of its physical properties and main chemical components. Different quality grades of tea have different market values; the higher the quality grade, the greater the market value. National tea grading standards generally rely on sensory evaluation, considering aspects such as appearance, color, taste, and purity. Existing research on the application of tea technology (shape, purity, color, aroma, and flavor) shows a close correlation between leaf tooth density and tea quality. Previous research on gene regulation of tea quality has mainly focused on the internal substances of tea; current research on the gene regulation of tea appearance is extremely limited. Based on this, this application is submitted. Summary of the Invention

[0003] The purpose of this invention is to provide tea trees CsTPR38 Application of genes in regulating leaf tooth density in tea plants CsTPR38 Genes can regulate leaf tooth density, laying the foundation for the breeding of new tea varieties.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of the tea plant CsTPR38 gene in regulating the leaf tooth density of tea plants, and the nucleotide sequence of the tea plant CsTPR38 gene is shown in SEQ ID No. 1.

[0005] Preferably, the CDS sequence of the tea plant CsTPR38 gene is shown in SEQ ID No. 2.

[0006] Preferably, the amino acid sequence of the tea plant CsTPR38 gene is shown in SEQ ID No. 3.

[0007] As a preferred method, inhibiting the expression of the CsTPR38 gene in tea plants significantly increases the number of leaf teeth.

[0008] As a preferred method, inhibiting the expression of the CsTPR38 gene in tea plants significantly increases the depth of leaf teeth.

[0009] This invention provides a recombinant plasmid for suppressing the expression of the CsTPR38 gene.

[0010] Preferably, the recombinant plasmid is pSH737-35S-CsTPR38-GUS.

[0011] This invention provides a method for regulating leaf tooth density in tea plants using the CsTPR38 gene, comprising the following steps: (1) The recombinant plasmid was introduced into Agrobacterium competent cells to obtain recombinant cells; (2) The recombinant cells were vacuum-stained and cultured on tea tree branches.

[0012] The present invention also provides the application of the recombinant plasmid in increasing the number of leaf teeth in tea leaves.

[0013] The present invention also provides the application of the recombinant plasmid in improving the leaf tooth depth of tea leaves.

[0014] This invention has the following technical effects and advantages: This invention, through the tea tree... CsTPR38 The sequence of the (tetratricopeptide repeat protein 38-like isoform X3) gene was cloned to obtain its complete sequence. Subsequently, bioinformatics analysis was performed. A plant expression vector was constructed, the target fragment was ligated to initiate GUS reporter gene expression, and the recombinant plant vector was used to genetically transform Arabidopsis thaliana, thereby verifying the gene's efficacy. CsTPR38 The function. For CsTPR38 The traits of the transgenic lines were analyzed, leaf tooth density was measured, and it was found that the tea plant... CsTPR38 Genes can regulate leaf tooth density, enabling the utilization of... CsTPR38 This lays the foundation for the genetic breeding of new varieties. Attached Figure Description

[0015] Figure 1 Silence for VIGS CsTPR38 The first leaf of the tea tree branch, from left to right: WT, pTRV2, pTRV2- CsTPR38 -1; Figure 2 Silence for VIGS CsTPR38 Number of serrations per leaf unit of the first leaf on a tea branch; Figure 3 Silence for VIGS CsTPR38 Depth of the first leaf tooth on a tea branch; Figure 4 Silence for VIGS CsTPR38 The first leaf of the tea tree branch CsTPR38 Relative gene expression levels; Figure 5The leaf teeth of the existing tea tree varieties (Yunwu Niaowang variety) are shown from left to right as Niaowang variety No. 2, Niaowang variety No. 10, Niaowang variety No. 30, Niaowang variety No. 9, and Niaowang variety No. 47, with a scale bar of 1000μm. Figure 6 The number of leaf teeth per unit leaf of the existing tea tree variety (Yunwu Bird King variety) and CsTPR38 Relative gene expression levels. Detailed Implementation

[0016] This invention provides the application of the tea plant CsTPR38 gene in regulating the leaf tooth density of tea plants. The nucleotide sequence of the tea plant CsTPR38 gene is shown in SEQ ID No. 1, and is as follows:

[0017] In this invention, the CDS sequence of the tea plant CsTPR38 gene is shown in SEQ ID No. 2, and is as follows:

[0018] In this invention, the amino acid sequence of the tea plant CsTPR38 gene is shown in SEQ ID No. 3, and is as follows: MKKLDRWGYEVNTSSDACISAINSYYHQVLTYGRDRSVILQAPVHDPHCVLANIFAAYFLSSSNPSRAPSHLQAAKARLLLKDFPRDLVSLKRVQVLCFYMGRSDLSL DLVQQVLPKNQQENYIYGMLAFPLLELGRMADAEEAAKKGFEINKEDSWAQHCLCHVLQYECRFKEAVEFMEECSTSWSSLSSFMYTHNWWHVALCYLEGHSSMRKVL EVYDNCIWKELERSDAVLPEVYLNAIGLLLRVYVRGEIDFFGDRLKILAGYLTNQAFWYLEWHLDVLILWALASTGAFDKGEDLKGLRFRISKMSKKKQQAMQSGML LAEAVYEYGKGNYKEALELLGPDFEANNFKTIGASDEQLDVFNEVWYVMLLNTGQAVKAIEVIGKRIKMREGAPFMWRLLERGYAMVGRPEATTVGEKAKYLETAYFK.

[0019] In this invention, inhibiting the expression of the CsTPR38 gene in tea plants significantly increases the number of leaf teeth.

[0020] In this invention, the expression of the CsTPR38 gene is suppressed in tea trees, and the leaf tooth depth of the tea trees is significantly increased. The tea tree variety is Wuniuzao.

[0021] This invention provides a recombinant plasmid for suppressing the expression of the CsTPR38 gene.

[0022] In this invention, the recombinant plasmid is pSH737-35S-CsTPR38-GUS.

[0023] This invention provides a method for regulating leaf tooth density in tea plants using the CsTPR38 gene, comprising the following steps: (1) The recombinant plasmid was introduced into Agrobacterium competent cells to obtain recombinant cells; (2) The recombinant cells were vacuum-impregnated onto tea tree branches and then cultured.

[0024] In this invention, the recombinant plasmid is introduced into Agrobacterium competent cells to obtain recombinant cells; preferably, the pTRV2-CsTPR38-GUS recombinant plasmid is introduced into GV3101 Agrobacterium competent cells to obtain recombinant cells pTRV2-CsTPR38.

[0025] In this invention, recombinant cells are vacuum-impregnated onto tea tree branches and then cultured. Preferably, the recombinant cells are mixed with a gene silencing vector, and after vacuum impregnation onto the tea tree branches, they are first cultured in the dark for 2-4 days, and then cultured in an artificial climate chamber for 30 days, with the culture medium changed every 7-15 days. The gene silencing vector is pTRV2, and the volume ratio of the recombinant cells to the gene silencing vector is 1:1. The preferred dark culture time is 3 days, and the tea tree branch variety is Wuniuzao.

[0026] The present invention also provides the application of the recombinant plasmid in increasing the number of leaf teeth in tea leaves.

[0027] The present invention also provides the application of the recombinant plasmid in improving the leaf tooth depth of tea leaves.

[0028] In this invention, the tea tree variety is Wuniuzao.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Main reagents used in the experiment

[0031] (1) YEP solid medium: 10 g / L peptone + 10 g / L yeast extract + 5 g / L sodium chloride + 7.5 g / L agar powder, pH=7.2; (2) YEP liquid culture medium: 10 g / L yeast extract + 10 g / L peptone + 5 g / L sodium chloride, pH=7.2; (3) Kanamycin (Kana) selection medium: 1 / 2 MS + 30 g / L sucrose + 50 mg / L kanamycin + 7.5 g / L agar; (4) Tea tree branch resuspension: 4.474 g / L MS, 30 g / L sucrose, 2 mg / L 6-BA, 0.1 mg / L NAA, 20 mg / L LAS, pH=5.8-6.0; (5) Tea tree branch culture medium: 0.2 mg / L 6-BA, 0.3 mg / L GA3, 25 mg / L chloramphenicol, pH=5.8-6.0; (6) LB liquid culture medium: 10g tryptone, 5g yeast extract, 10g NaCl, add ddH2O to make up to 1000mL, adjust pH to 7.0, autoclave at 121℃ for 20min; All reagents used were purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0032] In subsequent embodiments, the culture medium, resuspension solution and culture medium were all prepared in the manner described above.

[0033] Example 1

[0034] 1. Obtaining the target gene sequence

[0035] Data obtained from differences in the leaf tooth characteristics of ancient tea trees were analyzed using genome-wide association studies (GWAS) combined with tea tree genome databases. CsTPR38 (TEA025567.1) gene sequence.

[0036] 2. CsTPR38 Bioinformatics analysis of genes

[0037] The relevant functions of the CsTPR38 gene were predicted using online software (https: / / www.ncbi.nlm.nih.gov / orffinder / ; https: / / www.ebi.ac.uk / interpro / search / sequence / ).

[0038] 3.pTRV2- CsTPR38 -GUS recombinant plasmid (Changsha Kewen Biotechnology Co., Ltd.) introduced into Agrobacterium

[0039] (1) Take GV3101 Agrobacterium competent cells (stored at Guizhou University, volume 100μL) stored at -80℃ and thaw them on ice or at room temperature; (2) Under aseptic conditions, add 1 μg pTRV2- to the freshly thawed competent cell suspension. CsTPR38 -GUS (volume not exceeding 1 / 10 of the competent state volume), mix gently, and let stand in an ice water bath for 5 minutes; (3) Place the centrifuge tubes in liquid nitrogen for 5 minutes to freeze quickly; (4) Quickly place the centrifuge tube in a 28°C water bath for 5 minutes, and do not shake the cultured bacterial solution on the water surface; (5) Place the centrifuge tubes back into the ice water bath and keep them there for another 5 minutes; (6) Add 700 μL of antibiotic-free LB liquid medium under aseptic conditions and place it on a shaker at 28°C for 3 hours.

[0040] (7) After the culture is completed, centrifuge at 6000 rpm for 1 min, and leave about 100 μL of supernatant. Gently resuspend the bacterial cells by pipetting, and then add it to a YEP solid medium plate containing the corresponding antibiotic. Use a sterile bacterial spreader to spread the resuspended bacterial cells evenly. After the liquid in the plate is completely absorbed, invert the plate and place it in an incubator at 28°C for 2 days (if the plate contains 50 μg / mL Rif, it needs to be incubated at 28°C for 72 h; if the plate contains 50 μg / mL Kana and 20 μg / mL Rif, it needs to be incubated at 28°C for 48 h) until single colonies grow.

[0041] (8) Strain preservation: Pick a single colony and culture it overnight in 10 mL of YEP liquid medium containing 50 mg / L Rif and 50 mg / L Kana with shaking until OD 600 When the concentration reaches 1.0, add 500 μL of glycerol and 500 μL of bacterial solution to a 1.5 mL sterile centrifuge tube, mix gently, seal with sealing film and label, freeze quickly in liquid nitrogen, and then store in an ultra-low temperature freezer at -80°C.

[0042] Example 2: Virus-mediated tea tree branches CsTPR8 Gene silencing (VIGS)

[0043] 1. Agrobacterium activation and culture

[0044] (1) Take out the frozen bacterial culture (pTRV2-) obtained from the -80℃ freezer. CsTPR38 Agrobacterium strains pTRV2 and pTRV1 were obtained, with the specific method for obtaining pTRV2 and pTRV1 strains referring to Example 1). The bacterial suspension was thawed at room temperature. A loop was used to dip the bacterial suspension into YEP solid medium (containing 50 mg / L Kana and 50 mg / L Rif) and streaked onto the medium. The medium was then incubated upside down at 28°C for 2 days. (2) Pick a single colony and inoculate it into 10 mL of YEP liquid medium (containing 50 mg / L Kana and 50 mg / L Rif), and culture at 28°C and 200 rpm for 18 h with shaking. (3) Take 2 mL of the above bacterial culture and inoculate it into 250 mL of YEP liquid medium (containing 50 mg / L Kana, 50 mg / L Lf), and incubate on a shaker at 28°C and 200 rpm until OD. 600 Reached 1.2; (4) Centrifuge (4℃, 6000rpm, 10min), discard the supernatant, and resuspend in 250mL of resuspension (or with a final concentration OD of 1.2). Mix the resuspended pTRV2-CsTPR38 and pTRV1 Agrobacterium at a 1:1 volume ratio (experimental group), and mix pTRV2 and pTRV1 Agrobacterium at a 1:1 volume ratio (control group, pTRV2). After mixing, sonicate for 1min and activate by shaking on a shaker at 28℃ for 1h.

[0045] 2. Preparation and vacuum inoculation of plant materials

[0046] (1) Cut the branches (semi-lignified) of Wuniu early tea tree into small sections of 15-20cm with axillary buds, leaving 1-2 intact leaves.

[0047] (2) Place the prepared tea tree branches into a container of pTRV2- CsTPR38 Vacuum was drawn twice into a vacuum bottle containing a mixed culture of Agrobacterium pTRV1 (experimental group, or a mixed culture of Agrobacterium pTRV2 and pTRV1, control group). WT was vacuum-drawn directly using the resuspension.

[0048] (3) Remove the branches and thoroughly absorb the excess bacterial liquid from the branches and leaves.

[0049] (4) Place the branches in the culture solution and incubate in the dark for 3 days.

[0050] (5) After the dark preparation is completed, the culture medium is cultured in an artificial climate chamber for 30 days, and the culture medium is changed every 7 days.

[0051] The density and depth of leaf teeth in the new leaves of genetically modified tea tree branches, as well as the new leaves of genetically modified tea tree branches CsTPR38 The results of gene expression level determination are shown in [the table below]. Figures 1-4 .

[0052] Depend on Figures 1-3 It can be seen that pTRV2- CsTPR38 All strains had more leaf teeth per unit than the WT and pTRV2 strains, with strain 4 having the most. Except for strains 2 and 6, pTRV2- CsTPR38 Most strains have deeper leaf serrations than WT; except for strain 2, pTRV2- CsTPR38 Most strains have deeper leaf serrations than the pTRV2 strain. The leaf serrations of strain 1 are the deepest.

[0053] Depend on Figure 4 It can be seen that pTRV2- CsTPR38 All strains CsTPR38 The relative gene expression levels were significantly lower than those of the WT and pTRV2 lines.

[0054] Example 3

[0055] There were differences in leaf teeth among 108 individual Bird King tea trees selected and planted in the mother plant garden built in Yunwu Town, Guiding County. Figure 5 Five representative tea varieties (numbered 2, 9, 10, 30, and 47) show differences in the number of leaf serrations. CsTPR38 The relative expression levels of genes were measured, and the results are shown below. Figure 6 .

[0056] Depend on Figure 6 It can be seen that the number of serrations per unit leaf of tea leaf is inversely proportional to gene expression, which is consistent with gene silencing. CsTPR38 The results were consistent.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the tea plant CsTPR38 gene in regulating the leaf tooth density of tea plants, characterized by, The nucleotide sequence of the CsTPR38 gene of the tea plant is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The CDS sequence of the CsTPR38 gene of the tea plant is shown in SEQ ID No.

2.

3. The application according to claim 2, characterized in that, The amino acid sequence of the CsTPR38 gene of the tea plant is shown in SEQ ID No.

3.

4. The application according to claim 1, characterized in that, Inhibiting the expression of the CsTPR38 gene in tea plants significantly increased the number of leaf teeth.

5. The application according to claim 1, characterized in that, Inhibiting the expression of the CsTPR38 gene in tea plants significantly increases the depth of leaf teeth.

6. A recombinant plasmid, characterized in that, The recombinant plasmid was used to suppress the expression of the CsTPR38 gene.

7. The recombinant plasmid according to claim 6, characterized in that, The recombinant plasmid is pSH737-35S-CsTPR38-GUS.

8. A method for regulating leaf tooth density in tea plants using the CsTPR38 gene, characterized in that... Includes the following steps: (1) The recombinant plasmid according to claim 6 or 7 is introduced into Agrobacterium competent cells to obtain recombinant cells; (2) The recombinant cells were vacuum-stained and cultured on tea tree branches.

9. The application of the recombinant plasmid according to claim 6 or 7 in increasing the number of leaf teeth in tea trees.

10. The application of the recombinant plasmid according to claim 6 or 7 in improving the leaf tooth depth of tea leaves.

Images